This proposal focuses on human hereditary diseases that are caused by deficiencies in the glycolytic enzymes triosephosphate isomerase (TPI) or aldolase A. Deficiencies in either enzyme result in non-spherocytic hemolytic anemias. I propose to localize the molecular defects in TPI deficiency and aldolase a deficiency. TPI and aldolase genes will be isolated from phage libraries of normal human DNA. These genes will be structurally characterized and used as hybridization probes in experiments designed to analyze gene expression in normal fibroblast cell lines. RNA will be analyzed by S1 nuclease transcript mapping, RNA blotting and pulse-chase experiments, in combination with various cell fractionation procedures. Modifiers of glycolysis will be examined for an effect on TPI and aldolase gene regulation. Once the structure and expression of the normal genes have been determined, the exact molecular defect of several TPI- and aldolase A-deficient individuals will be investigated. Fibroblast cell lines established from deficient individuals will be analyzed for enzyme activity and immunologically cross-reacting material. These preliminary studies should differentiate between mutant alleles that generate protein with an altered primary structure and null alleles that do not produce any functional protein. Of these, null alleles are the more interesting and will be given priority in further analyses. Null alleles may be the result of mutations that affect gene transcription, RNA processing, mRNA stability, or protein stability. Mutant alleles will be isolated and sequenced, and fibroblast RNA that is transcribed from these alleles will be analyzed. Qualitative studies of mutant enzyme characteristics together with DNA sequence information and RNA metabolism will define the precise molecular lesion(s) in each mutant fibroblast cell line. Normal and defective gene regulation will also be examined in the mouse cell-bovine papilloma virus gene expression system. This system will eliminate any interpretative ambiguities that may result from studies with fibroblasts of affected individuals due to the presence of different alleles. In summary, TPI and aldolase A are two of many constitutively expressed genes which, as a group, remain uncharacterized in aspects of structure, regulatory sequences, and tissue-specific regulation. I will take advantage of naturally occurring mutations to determine the cause of several hereditary hemolytic anemias. This work on normal and mutant gene regulation will contribute to our understanding of how these "housekeeping" genes are expressed.